CN101333548A

CN101333548A - Method for preparing gamma-aminobutyric acid by lactobacillus brevis

Info

Publication number: CN101333548A
Application number: CNA2008101069494A
Authority: CN
Inventors: 曹郁生; 李海星
Original assignee: Nanchang University
Current assignee: Nanchang University
Priority date: 2008-06-26
Filing date: 2008-06-26
Publication date: 2008-12-31

Abstract

Disclosed is a method utilizing Lactobacillus brevis to prepare Gamma-aminobutyric acid, the technique comprises steps of: 1. utilizing an MRS liquid culture medium to activate the Lactobacillus brevis which is then inoculated into the MRSG fermentation culture medium by an inoculum size of 5 percent and cultivated for 40 to 60h at a temperature of 34DEG C for collecting thalli centrifugally at a temperature of 4DEG C; and 2. washing the culture medium for two times with sterilized physiological saline and hanging the culture medium in an acetic acid buffer containing 10-100mM of sodium glutamate and having a pH value of 5.2 for 1 to 8h for reaction at a temperature of 34DEG C, and obtaining a solution containing the Gamma-aminobutyric acid after centrifugation. The preparation method has the advantages of safe operation, low cost, mild reaction conditions, non environmental pollution and byproduct, and easy separation and purification of products.

Description

Utilize short lactobacillus to prepare the method for γ-An Jidingsuan

Technical field

The present invention relates to a kind of method for preparing γ-An Jidingsuan, relate in particular to a kind of method of utilizing short lactobacillus to prepare γ-An Jidingsuan.

Background technology

γ-An Jidingsuan is the natural amino acid that a kind of nonprotein is formed, it is a kind of important inhibitory nerve mediator in the mammalian body, have many important physical functions, as hypotensive, diuresis, stable, promote brain blood flow, trophic nerve cell, the sharp kidney of strong liver etc., and self imbalance of Woman climacteric had good improvement effect, can be used as medicine or healthcare products and use.

Milk-acid bacteria is that a class generally believes safe bacterium, is widely used in food-processing.Milk-acid bacteria has multiple physiologically active, and as regulating intestinal microflora, antibacterial anti-oxidant, strengthening immunity brings high blood pressure down etc.Japan has been widely used in γ-An Jidingsuan beverage, jam, cake, biscuit and food flavouring etc.The milk-acid bacteria that produces γ-An Jidingsuan not only produces γ-An Jidingsuan, and can keep the useful physiologically active of performance self, has a extensive future, and tangible economic benefit and social benefit are arranged.

Summary of the invention

The object of the present invention is to provide a kind of method of utilizing short lactobacillus to prepare γ-An Jidingsuan, this preparation method's safety, cost are low, the reaction conditions gentleness, and product is easy to separation and purification.

The present invention is achieved like this, and its processing method step is:

1. after utilizing the MRS liquid nutrient medium with the short lactobacillus activation, the inoculum size with 5% is inoculated in the MRSG fermention medium, cultivates 40-60h, 4 ℃ of centrifugal collection thalline for 34 ℃;

2. utilize 2 rear overhangs of stroke-physiological saline solution washing in the pH value that contains the 10-100mM Sodium Glutamate is 5.2 acetate buffer solution, 34 ℃ of reaction 1～8h are the solution that contains γ-An Jidingsuan after centrifugal.

The short lactobacillus that the present invention is used, deposit number is: Lactobacillus brevis CCTCCM208054.

MRS substratum among the present invention is: peptone 10g/L, beef extract 10g/L, yeast extract 5g/L, glucose 20g/L, K ₂HPO ₄2g/L, sodium acetate 5g/L, citric acid diamines 2g/L, MnSO ₄4H ₂O 0.25g/L, MgSO ₄7H ₂O 0.58g/L, tween 80 1mL, distilled water 1000mL through regulating pH6.2～6.4, sterilizes under high-temperature and high-pressure conditions.

MRSG substratum among the present invention is: peptone 10g/L, beef extract 10g/L, yeast extract 5g/L, glucose 20g/L, K ₂HPO ₄2g/L, sodium acetate 5g/L, citric acid diamines 2g/L, MnSO ₄4H ₂O 0.25g/L, MgSO ₄7H ₂O 0.58g/L, L-Sodium Glutamate 30g/L, tween 80 1mL, distilled water 1000mL through regulating pH6.2～6.4, sterilizes under high-temperature and high-pressure conditions.

Advantage of the present invention is: this method safety, cost are low, and the reaction conditions gentleness is free from environmental pollution, no coupling product, and product is easy to separation and purification.

Embodiment

Embodiment 1:

Be preserved in the short lactobacillus of MRS agar slant, transfer in the MRS liquid nutrient medium, after twice activation, the inoculum size with 5% is inoculated in the MRSG liquid nutrient medium, cultivates 48h in 34 ℃, the centrifugal 10min of 5000rpm, collecting precipitation utilizes aseptic physiological saline washing precipitation 2 times, at last precipitation is suspended from the acetate buffer solution (pH 5.2) that contains the 10mM Sodium Glutamate, 34 ℃ of reaction 1h are the solution that contains γ-An Jidingsuan after centrifugal.After measured, the Γ-aminobutyric acid in the fermented liquid reaches about 10mM.

Embodiment 2:

Be preserved in the short lactobacillus of MRS agar slant, transfer in the MRS liquid nutrient medium, after twice activation, the inoculum size with 5% is inoculated in the MRSG liquid nutrient medium, cultivates 48h in 34 ℃, the centrifugal 10min of 5000rpm, collecting precipitation utilizes aseptic physiological saline washing precipitation 2 times, at last precipitation is suspended from the acetate buffer solution (pH 5.2) that contains the 40mM Sodium Glutamate, 34 ℃ of reaction 2h are the solution that contains γ-An Jidingsuan after centrifugal.After measured, the Γ-aminobutyric acid in the fermented liquid reaches about 30mM.

Embodiment 3:

Be preserved in the short lactobacillus of MRS agar slant, transfer in the MRS liquid nutrient medium, after twice activation, the inoculum size with 5% is inoculated in the MRSG liquid nutrient medium, cultivates 48h in 34 ℃, the centrifugal 10min of 5000rpm, collecting precipitation utilizes aseptic physiological saline washing precipitation 2 times, at last precipitation is suspended from the acetate buffer solution (pH 5.2) that contains the 80mM Sodium Glutamate, 34 ℃ of reaction 4h are the solution that contains γ-An Jidingsuan after centrifugal.After measured, the γ-An Jidingsuan in the fermented liquid reaches about 60mM.

Embodiment 4:

Be preserved in the short lactobacillus of MRS agar slant, transfer in the MRS liquid nutrient medium, after twice activation, the inoculum size with 5% is inoculated in the MRSG liquid nutrient medium, cultivates 48h in 34 ℃, the centrifugal 10min of 5000rpm, collecting precipitation utilizes aseptic physiological saline washing precipitation 2 times, at last precipitation is suspended from the acetate buffer solution (pH 5.2) that contains the 100mM Sodium Glutamate, 34 ℃ of reaction 8h are the solution that contains γ-An Jidingsuan after centrifugal.After measured, the γ-An Jidingsuan in the fermented liquid reaches about 80mM.

MRS substratum in the foregoing description is: peptone 10g/L, beef extract 10g/L, yeast extract 5g/L, glucose 20g/L, K ₂HPO ₄2g/L, sodium acetate 5g/L, citric acid diamines 2g/L, MnSO ₄4H ₂O 0.25g/L, MgSO ₄7H ₂O 0.58g/L, tween 80 1mL, distilled water 1000mL through regulating pH6.2～6.4, sterilizes under high-temperature and high-pressure conditions.

MRSG substratum in the foregoing description is: peptone 10g/L, beef extract 10g/L, yeast extract 5g/L, glucose 20g/L, K ₂HPO ₄2g/L, sodium acetate 5g/L, citric acid diamines 2g/L, MnSO ₄4H ₂O 0.25g/L, MgSO ₄7H ₂O 0.58g/L, L-Sodium Glutamate 30g/L, tween 80 1mL, distilled water 1000mL through regulating pH6.2～6.4, sterilizes under high-temperature and high-pressure conditions.

The deposit number of short lactobacillus in the foregoing description is: Lactobacillus brevis CCTCCM208054.

Claims

1. method of utilizing short lactobacillus to prepare γ-An Jidingsuan is characterized in that the processing method step is:

(1) utilize the MRS liquid nutrient medium with after the short lactobacillus activation, the inoculum size with 5% is inoculated in the MRSG fermention medium, cultivates 40-60h, 4 ℃ of centrifugal collection thalline for 34 ℃;

(2) utilize 2 rear overhangs of stroke-physiological saline solution washing in the acetate buffer solution that contains 10-100mM Sodium Glutamate, pH 5.2,34 ℃ of reaction 1～8h are the solution that contains γ-An Jidingsuan after centrifugal.

2. a kind of method of utilizing short lactobacillus to prepare γ-An Jidingsuan according to claim 1, the deposit number of the short lactobacillus among the present invention is: Lactobacillus brevis CCTCCM 208054.

3. a kind of method of utilizing short lactobacillus to prepare γ-An Jidingsuan according to claim 1 is characterized in that the MRS substratum is: peptone 10g/L, beef extract 10g/L, yeast extract 5g/L, glucose 20g/L, K ₂HPO ₄2g/L, sodium acetate 5g/L, citric acid diamines 2g/L, MnSO ₄4H ₂O 0.25g/L, MgSO ₄7H ₂O 0.58g/L, tween 80 1mL, distilled water 1000mL through regulating pH6.2～6.4, sterilizes under high-temperature and high-pressure conditions.

4. a kind of method of utilizing short lactobacillus to prepare γ-An Jidingsuan according to claim 1 is characterized in that the MRSG substratum is: peptone 10g/L, beef extract 10g/L, yeast extract 5g/L, glucose 20g/L, K ₂HPO ₄2g/L, sodium acetate 5g/L, citric acid diamines 2g/L, MnSO ₄4H ₂O 0.25g/L, MgSO ₄7H ₂O 0.58g/L, L-Sodium Glutamate 30g/L, tween 80 1mL, distilled water 1000mL through regulating pH6.2～6.4, sterilizes under high-temperature and high-pressure conditions.